Transcription factors shape gene expression by binding to genomic cis-regulatory elements and then recruiting nucleosome remodeling factors, the RNA polymerase holoenyzme, and other transcriptional coactivators. What determines where a transcription factor binds in vivo? For most eukaryotic transcription factors (TFs), the answer to this question is not known. We have recently analyzed two yeast bHLH proteins, Cbf1 and Tye7, which have nearly identical DNA binding preferences in vitro, but bind at almost completely non-overlapping target loci in vivo. We found that Cbf1 utilizes homotypic cooperativity to achieve its specificity, while Tye7 binds in a TF collective, a phenomenon that has been described only recently in Drosophila, but is poorly understood. We hypothesize that homotypic cooperativity and collective binding are widely used by eukaryotic TFs to achieve their specificities in vivo. We will test this hypothesis by quantifying the contribution of these two mechanisms to the in vivo binding of all yeast transcription factors. We will also dissect a small number of these complexes in detail. We will investigate the binding specificity of the human bHLH transcription factor Usf1, which is a candidate drug target because of its involvement in obesity and metabolic disease. The completion of this work will deepen our understanding of the factors that govern the in vivo specificities of transcription factors. Furthermore, by gaining an understanding of the protein-protein contacts that regulate Usf1 binding, we will uncover interactions that can be disrupted for therapeutic benefit.